Abstract

The 2.4 MDa ribosome complex is responsible for protein synthesis in all domains
of life. During its biosynthesis, the nascent polypeptide chain (NC) threads through
the ribosomal exit tunnel and into the cellular milieu. There is much evidence to
indicate that during translation and whilst tethered to the ribosome, the NC has its
first opportunity to acquire structure, which assists in its folding to the active biological
state, in a process known as co-translational folding. The studies of the structural and
molecular determinants of this process present a challenge due to the NC’s intrinsic
conformational heterogeneity.
NMR spectroscopy has the unique ability to report on both protein structure and
dynamic at a residue-specific level and this thesis describes the development of NMR
methodologies to allow monitoring the progressive folding of an immunoglobulin
domain (ddFLN-dom5) NC as it emerges from the ribosome. Snapshots of the emergence
of ddFLN-dom5 from the ribosome were generated using different lengths of in vivo
translated, homogeneously stalled and selectively labelled ribosome-bound NCs (RNC)
which are then extracted from E. coli cells for NMR analysis. A strategy is described that
allows monitoring in situ the integrity of the RNC samples, and the attachment of the
NC to its parent ribosome.
Despite the high-molecular-weight of the ribosomal complexes, their instability and
the low achievable sample concentrations, a range of useful NMR tools are being
developed. Importantly, comparisons of 1H-13C methyl-TROSY HMQC and 1H-15N
SOFAST-HMQC NMR spectra of the ddFLN-dom5-RNCs with the isolated domain
in both native and denatured conditions allows the detailed analysis of the folding
equilibrium of the RNC. A robust data analysis methodology was designed to optimise
the significance of low signal to noise spectra. These NMR data reveal clear evidence
for co-translational folding when the C-terminal end of the ddFLN-dom5 is at lengths
47 residues from the peptidyl transferase centre (PTC). At this and longer linking
lengths, the chemical shifts observed for ddFLN-dom5-RNC are identical to those of the
isolated native domain. The RNC resonances show heterogeneous linewidth indicative
of conformational exchange between native and non-native states on the order of the
chemical-shift timescale (ms).
Overall, this study sets the stage for future opportunities for investigations of the
structural and dynamical properties of RNCs at a residue-specific level.